Method for producing indole derivative

ABSTRACT

The present invention provides a method for in vitro producing an indole derivative in a one-pot reaction. The method for producing a rhamnosylated indolocarbazole compound includes the steps of transforming a plasmid carrying a gene encoding N-glycosyltransferase into a bacterial strain; expressing the gene encoding N-glycosyltransferase in the bacterial strain; lysing the bacterial strain to obtain a crude enzyme extract; and adding TDP-glucose, an indolocarbazole aglycone and a metal ion in the crude enzyme extract for performing an enzymatic reaction to form the rhamnosylated indolocarbazole compound. Alternatively, the method for producing an indole-3-carboxaldehyde analog includes the steps of transforming a plasmid carrying a gene encoding NokA of  Nocardiopsis  sp. K-252 into a bacterial strain; expressing the gene encoding NokA in the bacterial strain; lysing the bacterial strain to obtain a crude enzyme extract; and adding an L-tryptophan analog for performing an enzymatic reaction to form the indole-3-carboxaldehyde analog.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for producing an indolederivative, and more particularly to a method for producing arhamnosylated indolocarbazole compound or an indole-3-carboxaldehydeanalog.

2. Description of Related Art

The family of indolocarbazole natural products has been a valuablesource of lead compounds with potential therapeutic applications in thetreatment of cancer and neurodegenerative disorders. In theindolocarbazole family, lestaurtinib has been approved by FDA fortreating acute leukemia, CEP-1347 has entered the phase III clinicaltrial for treating Parkinson's disease, and K-252a, K-252b andstaurosporine display anticancer activities. In addition, it is knownthat K-252d, rhamnosyl-K252c, is capable of inhibiting activity ofprotein kinase C (PKC). The PKC family plays an important role incellular proliferation and signal transduction. Hence, specificinhibitors against PKC are promising antitumor drugs for cancerchemotherapy.

S. Nakanishi et al. disclosed the extraction of K-252d from incubationmedium of a microorganism. (J. Antibiot., 1986, 39, 1066-1071) However,such extraction needs three purification steps to obtain only 13.3 mg ofK-252d from 8.4 L culture medium.

The synthesis of K-252d is summarized in the following Scheme I.

As shown in Scheme I, synthesis of TDP-rhamnose is accomplished bytandem enzymatic conversion of TDP-glucose with NDP-glucose4,6-dehydratase (Gdh), NDP-4-keto-6-deoxyglucose epimerase (Epi) andNDP-4-ketorhamnose reductase (Kre). Then, TDP-rhamnose is linked toK-252c by N-glycosyltransferase to form K-252d.

Chen et al. disclosed in vitro biosynthesis of TDP-rhamnose. (J. Biol.Chem., 2009, 284, 7352-7363) However, this method needs to purify threeenzymes, Gdh, Epi and Kre, and several processing steps.

In addition, it is known that indole-3-carboxaldehyde (ICA) hasanti-bacterial activity to gram positive bacteria such as S. aureus orto gram negative bacteria such as E. coli or E. faecium. Further, ICAcan be modified for treating stroke, cancer or neurodegeneration diseasesuch as Parkinson's disease. A modified ICA, 3-ICA-TSC, is an amebacide.

However, it is very complicated to purify ICA from a microorganism.(Nat. Prod. Res., 2005, 19, 645-652) S. S. Panda et al. disclosedchemical synthesis of ICA, which may cause environmental problems due tousage of organic solvents and toxic agents. (Indian J. Pharm. Sci.,2008, 70, 208-215)

Therefore, in order to overcome the drawbacks of the conventionalmethods, the present invention provide a novel method for in vitrosimply and efficiently producing an indole derivative.

SUMMARY OF THE INVENTION

The present invention provides a novel method for in vitro producing arhamnosylated indolocarbazole compound. The method includes the stepsof: transforming a plasmid carrying a gene encodingN-glycosyltransferase into a bacterial strain; expressing the geneencoding N-glycosyltransferase in the bacterial strain; lysing thebacterial strain to obtain a crude enzyme extract; and addingTDP-glucose, an indolocarbazole aglycone and a metal ion in the crudeenzyme extract for performing an enzymatic reaction to form therhamnosylated indolocarbazole compound.

Preferably, the bacterial strain is an E. coli strain, which ispreferably incubated at 25-40□.

Preferably, the N-glycosyltransferase is NokL of Nocardiopsis sp. K-252.

In accordance with the present invention, the step of lysing isperformed by a homogenizer (e.g. French press) or sonication.

In accordance with the present invention, the bacterial strain is lysedin a buffer selected from the group consisting of Tris buffer, HEPESbuffer, MOPS buffer, K₂HPO₄ buffer and MES buffer. Preferably, pH of thebuffer is in a range from 5 to 10, and the buffer contains 0-25% ofglycerol.

In accordance with the present invention, the indolocarbazole aglyconeis a compound of formula (I) or a compound of formula (II), or itsanalog

in which R═H, OH, F, Cl, Br or CH₃, and wherein the rhamnosylatedindolocarbazole compound is K-252d (or its analogs).

In accordance with the present invention, the metal ion is a magnesiumion or a manganese ion, and the enzymatic reaction is performed at4-40□.

In addition, the present invention provides a method for in vitroproducing an indole-3-carboxaldehyde analog. The method includes thesteps of: transforming a plasmid carrying a gene encoding NokA ofNocardiopsis sp. K-252 into an E. coli strain; expressing the geneencoding NokA in the E. coli strain; lysing the E. coli strain to obtaina crude enzyme extract; and adding an L-tryptophan analog of formula(III)

wherein R═H, OH, F, Cl, Br or CH₃. Preferably, the E. coli strain islysed in a buffer selected from the group consisting of Tris buffer,HEPES buffer, MOPS buffer, K₂HPO₄ buffer and MES buffer, wherein pH ofthe buffer is in a range from 5 to 10, and the buffer contains 0-25% ofglycerol.

In the following section preferred embodiments are described. However,this is not intended in any way to limit the scope of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more fully understood by reading thefollowing detailed description of the preferred embodiments, withreference made to the accompanying drawings, wherein:

FIG. 1 is a construct map of pCY16 according to the present invention;

FIG. 2 is a scheme showing synthesis of K-252d according the embodimentof the present invention;

FIG. 3 is a construct map of pJZ22 according to the present invention;and

FIG. 4 is a scheme showing synthesis of indole-3-carboxaldehyde analogsaccording the embodiment of the present invention;

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following illustrative embodiments are provided to illustrate thedisclosure of the present invention, these advantages and effects can beapparently understood by those in the art after reading the disclosureof this specification. The present invention can also be performed orapplied by other different embodiments. The details of the specificationmay be on the basis of different points and applications, and numerousmodifications and variations can be devised without departing from thespirit of the present invention.

It is known that Nocardiopsis sp. K-252 (Nonomuraea longicatena K252T,NRRL15532) produces indolocarbazole alkaloids of antitumor antibiotics.Thus, the inventors constructed a fosmid genomic DNA library ofNocardiopsis sp. K-252 by using a CopyControl fosmid library productionkit (Epicentre). As a result, the genomic library was constructed with atotal of 5856 fosmid clones, whereas the average sizes of genomic DNAfragments were ca. 35 kb per clone. A 45 kb sequence contig wassubsequently obtained by DNA sequencing to cover the entire gene clusterfor the biosynthesis of the indolocarbazole compounds, K-252a and itsanalogs, in Nocardiopsis sp. K-252. The DNA sequence of nok genesresponsible for biosynthesis of K-252a was deposited in GenBank underaccession number FJ031030. Sequence analysis of the 45 kb genomicsequence revealed 35 open reading frames. The inventors identified thegene nokL (SEQ ID NO: 1; GenBank accession number: ACN29718) encodingN-glycosyltransferase, and the gene nokA (SEQ ID NO: 2; GenBankaccession number: ACN29719) encoding L-amino acid oxidase. The detaileddescription about molecular cloning, sequence analysis and functionalcharacterization of the gene cluster for biosynthesis of K-252a and itsanalogs has been published on Mol. BioSyst., 2009, 5, 1180-1191, whichis entirely incorporated herein by reference.

The present invention provides a heterologous expression system ofEscherichia coli containing indolocarbazole N-glycosyltransferase for invitro producing molecules exhibiting potent neuroprotective or broadanticancer activities in a one-pot reaction.

In the present invention, the plasmid containing the DNA encodingN-glycosyltransferase is transformed into an E. coli strain, which isthen incubated until OD₆₀₀ being 0.3-0.7. Preferably, theN-glycosyltransferase is NokL of Nocardiopsis sp. K-252. After adding aninducing agent, the bacterial culture is further incubated at a 25-40□.Then, the culture pellet is collected and further re-suspended in abuffer solution to be lyzed by a homogenizer (e.g. French press) orsonication, such that the crude enzyme extract is obtained. Preferably,the buffer solution is Tris, HEPES, MOPS, K₂HPO₄ or MES, the pH value ofthe buffer solution is in the range from 5 to 10, and the glycerolconcentration of the buffer solution is 0-25%.

Subsequently, the crude enzyme extract is mixed with TDP-glucose,indolocarbazole aglycone, K-252c or its analog, and metal ions. Theindolocarbazole aglycone is the compound of formula (I) or the compoundof formula (II), in which R═H, OH, F, Cl, Br or CH₃. The metal ions aremagnesium ions or manganese ions. The enzymatic biosynthesis isperformed at 4-40° C.

In addition, the present invention provides a method for in vitroproducing ICA and its analogs in a one-pot reaction. In the presentinvention, the plasmid containing the DNA encoding L-amino acid oxidaseis transformed into an E. coli strain, which is then incubated untilOD₆₀₀ being 0.3-0.7. Preferably, the L-amino acid oxidase is NokA ofNocardiopsis sp. K-252. After adding an inducing agent, the bacterialculture is further incubated at a 25-40° C. Then, the culture pellet iscollected and further re-suspended in a buffer solution to be lyzed by ahomogenizer (e.g. French press) or sonication, such that the crudeenzyme extract is obtained. Preferably, the buffer solution is Tris,HEPES, MOPS, K₂HPO₄ or MES, the pH value of the buffer solution is inthe range from 5 to 10, and the glycerol concentration of the buffersolution is 0-25%.

Subsequently, the crude enzyme extract is mixed with an L-tryptophananalog of formula (III), wherein R═H, OH, F, Cl, Br or CH₃. Theenzymatic biosynthesis is performed at 4-40□.

Biochemical characterization and substrate specificity of the genecluster for biosynthesis of K-252a and its analogs by in vitroheterologous expression system of Escherichia coli has been published onMol. BioSyst., 2009, 5, 1192-1203, which is entirely incorporated hereinby reference.

Embodiment of In Vitro Biosynthesis of K252d:

Construction of the NokL Expression Plasmid

FIG. 1 shows the construct map of pCY16. The gene nokL (SEQ ID NO: 1) ofNocardiopsis sp. K-252 was amplified on pJC3B5 by PCR with a forwardprimer with an NdeI site and a reverse primer with a stop codon (TGA)followed by an EcoRI site near 5′-end. The amplified PCR product wasligated with blunt-ended pUC19 (NEB) at SmaI to generate pCY15. Afterdigestion of pCY15 with NdeI and EcoRI, the digestion fragment carryingnokL was cloned into pET21b vector to give pCY16 for the wild-type NokLexpression experiments. For plasmid construction of N-terminalHis₆-tagged NokL, the same digestion fragment was cloned into pET28a toafford pCY17. For C-terminal His₆-tagged NokL expression, the nokL genewas amplified on pCY16 by PCR with primer pairs of NKLNdF1 (forward,with NdeI) and NKLXR1 (reverse, with XhoI). The resulting PCR productwas subsequently cloned into pET21b at the corresponding sites to yieldpMS4.

Preparation of the NokL Cell-Free Crude Extract

The pCY 16 (NokL) and pG-KJE7 (chaperones) plasmids were co-transformedinto E. coli BL21 (DE3). Cells were grown at 37° C. in LB medium withantibiotics (100 μg/ml ampicillin and 30 μg/ml kanamycin) until 0D₆₀₀reached 0.5. After induction with 0.1% (w/v) L-arabinose and 1 mM IPTG,the culture was allowed to grow at 300 for additional 20 hours. Allprocedures for the preparation of cell-free crude extract were carriedout on ice or at 40. The cells were harvested by centrifugation (3200 g,15 min), followed by resuspension with potassium phosphate buffer (20 mMK₂HPO₄, pH 7.8, 15% glycerol). Cells were broken and disrupted by twopassages through a French press cell (Spectronic Instruments) at 16 000psi. After removal of cell debris by centrifugation at 16000 g for 20min, the desired crude NokL enzyme extract was obtained for thefollowing enzymatic reaction.

Production of K-252d from Cell-Free Enzymatic Reaction

The crude NokL enzyme extract (1 mL, 20 mM K₂HPO₄ at pH 7.8, 15%glycerol) was mixed with TDP-glucose (1.5 mM, 1 mL, 12 mM Tris-HCl,pH7.6, 9% glycerol), K-252c (1.27 mM, 1 mL, 50% DMSO), MgCl₂ (12 mM, 1mL in H₂O), and then the pH of the mixture was adjusted to 9.0 (12 mMMgCl₂, 50 mM K₂HPO₄). The reaction of the mixture was performed at 30□for 24 hours.

Identification of NokL Enzymatic Product (K252d)

After the enzymatic reaction, the above reaction mixture was

quenched by 5 ml of the ice-cold alcohol solution (MeOH-EtOH). Theresulting mixture was subsequently subjected to centrifugation (16,000g, 4□) for 2 hours to remove precipitated proteins. The supernatant waspurified by semi-preparative RP-HPLC. Fractions containing K252d werepooled and evaporated to remove organic solvents, followed by extractionwith ether. The ether layer containing K252d was evaporated to removeether and then vacuumed to gain the K252d (2.0 mg). The purity of K252dwas greater than 95% as judged by analytical RP-HPLC. The NMR spectrumof K252d dissolved in D₄-methanol (CD₃OD) was recorded at 500 MHz. ForNMR analysis: ¹H-NMR (CD₃OD, 500 MHz) δ_(H) 1.80 (3H, d, J=7.0 Hz), 4.15(1H, dd, J=4.0 Hz), 4.33 (1H, td, J=4.0 Hz), 4.55 (1H, m), 4.70 (1H, d,J=4.0 Hz) 5.05 (2H, d, J=4.0 Hz), 6.54 (1H, d, J=10.0 Hz), 7.26 (1H, t,J=7.0 Hz), 7.30 (1H, t, J=8.5 Hz), 7.45 (1H, t, J=8.0 Hz), 7.48 (1H, t,J=8.5 Hz), 7.61 (1H, d, J=8.5 Hz), 7.72 (1H, d, J=8.5 Hz), 8.02 (1H, d,J=8.5 Hz), 9.40 (1H, d, J=8.0 Hz) ppm. ¹³C-NMR (CD₃OD, 125 MHz) δ_(C)15.8, 47.0, 68.7, 73.3, 73.6, 78.5, 78.6, 110.5, 112.3, 116.5, 118.9,119.7, 120.7, 121.0, 121.9, 123.6, 124.1, 126.2, 126.5, 126.8, 129.0,129.7, 134.5, 141.0, 142.2, 175.8 ppm. For high resolution MALDI-TOFspectrometric analysis: C₂₆H₂₃N₃O₅ molecular weight calculated as457.163, and found m/z of 457.177 [M]⁺. Upon ¹H-NMR, ¹³C-NMR and COSY,the rhamnosylated product was fully assigned with chemical shifts, inexcellent agreement with those of K-252d reported by Yasuzawa (J.Antibiot., 1986, 39, 1072-1078).

As summarized in the scheme of FIG. 2, the present invention provides anovel method for in vitro producing K-252d in a one-pot reaction withoutpurifying any enzyme. Furthermore, the method of the present inventioncan produce various indolocarbazole derivatives by using variousindolocarbazole aglycones.

Embodiment of In Vitro Biosynthesis of indole-3-carboxaldehyde:

Construction of the NokA Expression Plasmid

FIG. 3 shows the construct map of pJZ22. The gene nokA (SEQ ID NO: 2) ofNocardiopsis sp. K-252 was amplified on pJC3B5 by PCR with a primer pairwith NdeI and NheI sites at the 5′ and 3′ ends, respectively. Theamplified PCR product preserving the stop codon was cloned into the NdeIand NheI sites of pET21b to generate pJZ22.

Preparation of the Noka Cell-Free Crude Extract

The pJZ22 (NokA) and pG-KJE7 (chaperones) plasmids were co-transformedinto E. coli BL21 (DE3). Cells were grown at 37□ in LB medium withantibiotics (100 μg/ml ampicillin and 30 μg/ml kanamycin) until OD₆₀₀reached 0.5. After induction with 0.2% (w/v) L-arabinose and 0.25 mMIPTG, the culture was allowed to grow at 30□ for additional 10 hours.All procedures for the preparation of cell-free crude extract werecarried out on ice or at 4□. The cells were harvested by centrifugation(1902 g, 20 min), followed by resuspension with the buffer (104 mMTris-HCl, pH 7.6, 10% glycerol). Cells were broken and disrupted bysonication. After removal of cell debris by centrifugation at 15700 gfor 20 min, the desired crude NokA enzyme extract was obtained for thefollowing enzymatic reaction.

Production of indole-3-carboxaldehyde from Cell-Free Enzymatic Reaction

The crude NokA enzyme extract (80 μl, 80 mM Tris-HCl at pH 7.8, 7.6%(v/v) glycerol) was mixed with L-tryptophan (4 mM), and then incubatedin a total volume of 104 μl at 30□ for 24 hours.

Identification of NokA Enzymatic Product (indole-3-carboxaldehyde)

After the enzymatic reaction, the above reaction mixture was quenched byan equal volume of ice-cold MeOH, and was then subjected to reversephase RP-HPLC analysis using by Agilent 1100 HPLC series equipped withquaternary pump and diode-array detector. As a result, RP-HPLC analysisof the NokA reaction revealed a major product peak and two minor ones.Upon characterization by NMR and mass spectroscopy, the major productwas found to be indole-3-carboxaldehyde (ICA). ¹H-NMR (CD₃OD, 500 MHz),δ_(H) 7.170 (1H, ddd, J=1, 7.5 Hz), 7.212 (1H, ddd, J=1.5, 8 Hz), 7.410(1H, d, J=8 Hz), 8.031 (1H, s), 8.091 (1H, d, J=8 Hz), and 9.822 (1H, s)ppm. ¹³C-NMR (CD₃OD, 125 MHz), δ_(C) 113.122, 120.132, 122.384, 123.611,124.998, 125.722, 138.940, 139.673, and 187.406 ppm.

As summarized in the scheme of FIG. 4, the present invention provides anovel method for in vitro producing ICA derivatives in a one-potreaction without purifying any enzyme.

The invention has been described using the exemplary preferredembodiment. However, it is to be understood that the scope of theinvention is not limited to the disclosed arrangements. The scope of theclaims, therefore, should be accorded the broadest interpretation, so asto encompass all such modifications and similar arrangements.

1. A method for in vitro producing a rhamnosylated indolocarbazolecompound, comprising the steps of: transforming a plasmid carrying agene encoding N-glycosyltransferase into a bacterial strain; expressingthe gene encoding N-glycosyltransferase in the bacterial strain; lysingthe bacterial strain to obtain a crude enzyme extract; and addingTDP-glucose, an indolocarbazole aglycone and a metal ion in the crudeenzyme extract for performing an enzymatic reaction to form therhamnosylated indolocarbazole compound.
 2. The method of claim 1,wherein the bacterial strain is an E. coli strain.
 3. The method ofclaim 2, wherein the E. coli strain is incubated at 25-40° C.
 4. Themethod of claim 1, where in the N-glycosyltransferase is NokL ofNocardiopsis sp. K-252.
 5. The method of claim 1, wherein the step oflysing is performed by a homogenizer or sonication.
 6. The method ofclaim 1, wherein the bacterial strain is lysed in a buffer selected fromthe group consisting of Tris buffer, HEPES buffer, MOPS buffer, K₂HPO₄and MES buffer.
 7. The method of claim 6, wherein pH of the buffer is ina range from 5 to
 10. 8. The method of claim 6, wherein the buffercontains 0-25% of glycerol.
 9. The method of claim 1, wherein theindolocarbazole aglycone is a compound of formula (I) or a compound offormula (II), or its analog

in which R═H, OH, F, Cl, Br or CH₃, and wherein the rhamnosylatedindolocarbazole compound is K-252d or its analogs.
 10. The method ofclaim 1, wherein the metal ion is a magnesium ion or a manganese ion.11. The method of claim 1, wherein the enzymatic reaction is performedat 4-40° C.
 12. A method for in vitro producing anindole-3-carboxaldehyde analog, comprising the steps of: transforming aplasmid carrying a gene encoding NokA of Nocardiopsis sp. K-252 into abacterial strain; expressing the gene encoding NokA in the bacterialstrain; lysing the bacterial strain to obtain a crude enzyme extract;and adding an L-tryptophan analog of formula (III) in the crude enzymeextract

wherein R═H, OH, F, Cl, Br or CH₃, for performing an enzymatic reactionto form the indole-3-carboxaldehyde analog.
 13. The method of claim 12,wherein the bacterial strain is an E. coli strain.
 14. The method ofclaim 13, wherein the E. coli strain is incubated at 25-40° C.
 15. Themethod of claim 13, wherein the E. coli strain is lysed in a bufferselected from the group consisting of Tris buffer, HEPES buffer, MOPSbuffer, K₂HPO₄ buffer and MES buffer.
 16. The method of claim 15,wherein pH of the buffer is in a range from 5 to
 10. 17. The method ofclaim 15, wherein the buffer contains 0-25% of glycerol.
 18. The methodof claim 12, wherein the step of lysing is performed by a homogenizer orsonication.
 19. The method of claim 12, wherein the enzymatic reactionis performed at 4-40° C.